Pyrazolopyrimidines are a general class of compounds which exhibit Aj adenosine receptor affmity. A number of pyrazolo(3,4-d)pyrimidine analogues of isoguanosine and i-methylisoguanosine has been synthesised. All compounds were tested forAi adenosine receptor affinity using a (311) R-PIA competitive binding assay. The N-i and N-5 positions were substituted with a number of different ailcyl and aryi groups. 3-Chiorophenyl substitution of the N-i position and butyl substitution of the N-5 position greatly enhanced the overall adenosine receptor affinity. Substitution by a methyl group at the N-7 position fixed the C-4 position in the imino tautomeric form. This resulted in a marked reduction in activity. The substitution of the N-2 position with a phenyl group produced an analogue with a similar structure to i,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX). A 2-phenyl substituent was favourable for interaction with the adenosine receptor. A number of pyrazolo(3,4-d)pyrirnidine analogues of 4,6-bis-a-carbamoylethylthio-i-phenylthiopyrazolo(3,4-d)pyrinhidine (DJB-KK) has also been synthesised and tested for Aj adenosine receptor affinity. 4,6-Bis-alkylthio-1-phenylpyrazolo(3,4-d)pyrimidines with a-carbamoylethyl and u-carbamoylpropyi groups were compared. The additional methyiene of the a-carbamoylpropyl group produced increased adenosine receptor affinity. 6-a-Carbamoylethylthio-4-mercapto-1-phenylpyrazolo(3,4-d)pyrimidine and 4-cc-carbamoylethylthio- i-phenylpyrazolo(3,4-dlpyrimidine were compared. Substitution of the C-6 position maintained activity, while substitution of the C-4 reduced activity.

The difficulty with movement initiation, or akinesia, is a cardinal symptom of Parkinson�s disease (PD) and the loss of dopaminergic cells, affecting the function of the basal ganglia, the thalamus and the motor cortex, has long been documented. From a broader perspective, it has been proposed that akinesia is caused by impaired function in different brain areas, inside and outside the basal ganglia, operating as a �behavioural arrest control system� (Klemm, 2001). Several neurotransmitters seem to modulate the activity of this system and, contrasting the well-known effects of dopamine, the involvement of adenosine has only recently emerged, particularly via A2A receptors. Adenosine plays an opposite role to dopamine in the brain: adenosine stimulation at A2A receptors inhibits movement (Ferre et al., 1991a; Hauber and Munkle, 1995; Rimondini et al., 1997), whereas A2A antagonists seem to promote movement (Kanda et al., 2000; Bara-Jimenez et al., 2003; Pinna et al., 2005). Although specific adenosine A2A and dopamine D2 receptors are known to antagonistically interact (Ferre et al., 1997; Fuxe et al., 1998; Ferre et al., 2001), little is known of the involvement of A2A receptors in regulating neural activity in the basal ganglia, a crucial point for the future use of A2A antagonists as adjuvant therapy in Parkinson�s disease. In fact, although it is generally accepted that akinesia results from altered function in the cortico-basal ganglia-cortical loop, as confirmed in several studies reporting changes in basal ganglia activity following dopamine depletion (Blandini et al., 2000; Bevan et al., 2002; Boraud et al., 2002), no study to date has systematically investigated electrophysiological changes in the basal ganglia during akinesia induced by adenosine receptor stimulation. Starting from a common behavioural effect, this study tries to bridge this gap by investigating and comparing, in two basal ganglia structures, the neural substrate of akinesia after acute dopamine D2 receptor blockade and adenosine A2A receptor stimulation. The external segment of the globus pallidus (GP, or simply globus pallidus in the rat) and the substantia nigra pars reticulata (SNr) were chosen as the recording sites because both nuclei are included into the �behavioural arrest control system� and seem to express somewhat complementary functions, as a respective key integrative station and main output of the basal ganglia. Dopamine function was manipulated by acute decrease in availability of dopamine binding sites in the brain, through specific dopamine D2 receptor blockade with systemic injections (1.0 and 1.5 mg/kg) of raclopride(3,5-dichloro-N-[(1-ethylpyrrolidin-2-y)methyl]-2-hydroxy-6-methoxy-benzamide), resulting in akinesia. Conversely, movement was inhibited by specific adenosine A2A receptor stimulation with systemic injections (2.5 and 5.0 mg/kg) of the drug CGS21680 (sodium-2-p-carboxyethylphenylamino-5-N-carboxamidoadenosine). In both situations, behaviour was assessed through specific akinesia tests. Single neuron activity before injection and changes in the firing frequency and firing pattern occurring after injection have been analysed and compared for each cell recorded from GP and SNr, during periods of behavioural rest. Synchronised firing between cell pairs has also been assessed. However, the small number of cell pairs showing correlated firing in each structure after systemic injection of drugs was not statistically relevant for further analysis and interpretation of synchronised firing during drug induced akinesia. In our experiments, both drugs inhibited movement, albeit somewhat differently, with lack of rigidity and �flat� body position after adenosine stimulation. Dopamine blockade decreased mean firing rate and dramatically altered the firing pattern in both investigated structures, generally increasing burst activity (increased percentage of spikes in bursts, mean number of bursts, mean number of spikes per burst, mean intra-burst firing frequency) and decreasing regularity of firing (increased coefficient of variation of the inter-spike intervals). Increased burst activity in the rat basal ganglia in an acute model of parkinsonian akinesia, following systemic raclopride injections, confirmed the importance of changes in the firing pattern in PD. The only electrophysiological effect of systemic A2A stimulation was decreased mean firing rate in the GP, a weak effect that could not propagate towards output stations of the basal ganglia. The lack of changes in the firing pattern, at both input and output levels of the basal ganglia, suggests a correlation with the lack of rigidity in adenosine-stimulation-induced akinesia.

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Graduation date: 1990

The role of inhibitory compounds such as adenosine and GABA in modulating neuronal activity in invertebrate species is not well described. Here I investigate their role in modulating excitability of cluster F neurons in the pedal ganglia of Lymnaea stagnalis. Receptor-specific agonists and antagonists were used to determine that the inhibitory effects of adenosine were mediated through the adenosine A1 receptor, and that action potential frequency varied linearly with intracellular calcium concentrations. These effects had a seasonal dependence, as neurons were resistant to adenosinergic modulation during the summer months. GABAergic modulation of neuronal activity was also seasonal as demonstrated by ionic plasticity in GABAergic transmission. GABA application led to inhibition or excitation of electrical activity in neurons obtained during the fall and winter months, respectively. These effects were mediated through the GABA(A) receptor because of sensitivity to GABA(A) receptor antagonist bicuculline and were likely due to differential cation-chloride cotransporter activity.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

The role of inhibitory compounds such as adenosine and GABA in modulating neuronal activity in invertebrate species is not well described. Here I investigate their role in modulating excitability of cluster F neurons in the pedal ganglia of Lymnaea stagnalis. Receptor-specific agonists and antagonists were used to determine that the inhibitory effects of adenosine were mediated through the adenosine A1 receptor, and that action potential frequency varied linearly with intracellular calcium concentrations. These effects had a seasonal dependence, as neurons were resistant to adenosinergic modulation during the summer months. GABAergic modulation of neuronal activity was also seasonal as demonstrated by ionic plasticity in GABAergic transmission. GABA application led to inhibition or excitation of electrical activity in neurons obtained during the fall and winter months, respectively. These effects were mediated through the GABA(A) receptor because of sensitivity to GABA(A) receptor antagonist bicuculline and were likely due to differential cation-chloride cotransporter activity.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

The hydrolysis of adenosine, 2-deoxyadenosine and 6-chloropurine riboside by adenosine deaminase preparations from human spleen has been investigated, and Km and Vm values have been determined. The effect of the substrate on the reaction velocity was followed over a 250-fold range Results showed no deviation from Michaelis-Menten kinetics. The effect of pH on Vm, Vm/Km, and the apparent activation energy was examined. Inactivation of the enzyme by p-chloromercuribenzoate suggests the involvement of one or more SH groups. Competitive inhibition by inosine and the fact that ammonia is not, an inhibitor support a reaction mechanism involving a ternary complex. The apparent activation energy of the a parameter was smaller or less sensitive to temperature than the ß parameter.Ball State UniversityMuncie, IN 47306

Carbon monoxide (CO) has been identified as an endogenous biological messenger in the brain. Heme oxygenase (HO) catalyzes the metabolism of heme to CO and biliverdin. CO has been shown to act as a neurotransmitter and neuronal messenger in the brain. We reported recently that CO was involved in central cardiovascular regulation, modulated the baroreflex, may affect glutamatergic neurotransmission, and metabotropic glutamate receptors (mGluRs) may be coupled to the activation of HO in the nucleus tractus solitarii (NTS) of rats. We also reported previously that adenosine can increase the release of glutamate in the NTS. The present study was designed to investigate the possible interaction of CO, adenosine, and mGluRs groups in the NTS. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure were monitored intra-arterially. Unilateral microinjection of ascending doses of hemin (0.01 to 3.3 nmol), a heme molecule cleaved by HO to yield CO, produced decreases in blood pressure and heart rate dose-dependently. In addition, similar cardiovascular effects were observed in adenosine (2.3 nmol) and several agonists for mGluRs groups such as DHPG (group ¢¹) (0.03 nmol), APDC (group ¢º) (0.3 nmol)and L-AP4 (group ¢») (0.3 nmol). These cardiovascular effects of hemin were attenuatd by prior administration of the adenosine receptor antagonist DPSPX (0.92 nmol). Similarly, pre-treatment of HO inhibitor ZnPP¢Á or ZnDPBG (1 nmol) also attenuated the depressor and bradycadic effects of adenosine. Among the mGluRs agonists, prior administration of ZnPP¢Á (1 nmol), an inhibitor of HO activity, significantly attenuated the cardiovascular effects of APDC and L-AP4, and failed to prevent the cardiovascular responses of DHPG. These results indicated an interaction between CO and adenosine, and group ¢º and ¢» mGluRs may be coupled to the activation of HO in central cardiovascular regulation.